This application claims priority under 35 U.S.C. xc2xa7xc2xa7119 and/or 365 to Patent Application No. 97-04954 filed in France on Apr. 22, 1997; the entire content of which is hereby incorporated by reference.
The present invention relates to novel nucleic acid sequences which can be involved in controlling and regulating the expression of genes encoding MHC type II molecules and to their use, in particular as drugs for treating disorders in which it is desirable to act on the level at which genes encoding MHC type II molecules are expressed.
The molecules of the class II major histo-compatibility complex (termed MHC in that which follows) are heterodimeric transmembrane glycoproteins which are directly involved in activating T helper CD4+ lymphocytes during the course of the immune response.
In man, this class II complex is represented by the molecules which belong to the HLA (human leucocyte antigen) system. The genes which encode the xcex1 and xcex2 chains of which the HLA-DR, HLA-DQ and HLA-DP molecules are composed are located within the D region of chromosome 6.
Expression of these genes is very highly regulated. In contrast to the genes which encode the MHC type I molecules, which are expressed ubiquitously, expression of the genes which encode the MHC class II molecules is either constitutive, in only a few cell types such as B lymphocytes, activated T lymphocytes, macrophages, cells of the thymic epithelium, or dendritic cells such as the Langerhans cells, or is induced following stimulation, for example by cytokines, more specifically by interferon xcex3 (INF xcex3) or interleukin 4 (IL4), in several other cell types such as cells which belong to the macrophage or monocyte line, endothelial cells, fibroblasts, muscle cells or cancer cells such as melanoma cells.
Furthermore, expression of the genes which encode MHC class II molecules in B lymphocytes is transient. Thus, differentiation of the B cells into plasma cells which produce the immunoglobulins is accompanied by the suppression of certain genes including those which encode MHC class II.
Similarly, it has been shown that the level at which MHC type II molecules are expressed is a determining factor in the process of T cell activation.
As a consequence, it is clearly apparent that the molecular mechanisms by which expression of these genes is regulated constitute a key element in the efficacy of the immune response. Any defect in this regulatory process may result in significant immunological disorders or autoimmune diseases. Thus, abnormal expression of the MHC class II genes has in some cases been observed at the surface of cells which should not normally express these genes. Similarly, it is possible to observe over-expression of these genes, leading to an activation of the CD4+ lymphocytes which is aberrant and uncontrolled [Bottazzo et al., 1986, Immunol. Rev., 94, 137-169]. Events of this kind could, at least in part, be responsible for disorders such as insulin-dependent diabetes, multiple sclerosis, rheumatoid arthritis and lupus erythematosus. Conversely, it has been possible to demonstrate an immunodeficiency in some patients which has resulted from a disturbance in the expression of MHC class II genes. Mention may, for example, be made of the BLS (bare lymphocytes syndrome) syndrome which is a recessive autosomal disorder in which expression of the MHC class II genes is very limited if not to say non-existent, a situation which finds expression in the absence of cellular and humoral immune responses and is accompanied by a large number of infections which are often fatal.
Several scientific groups have analysed the mechanisms by which expression of the MHC class II genes is regulated and have identified a number of transactivating molecules which are capable of binding, directly or indirectly, to promoter sequences which are specific for the said genes [for a review, see Mach et al., 1996, Annu. Rev. Immunol. 14, 301-331].
The applicant has previously identified and characterized one of these factors, i.e. the CIITA factor (class II transactivator) [Steimle et al., 1993, Cell 75, 135-146 and EP 648836]. Furthermore, document WO 9606107 shows that there are two domains within the CIITA factor which are more involved in activating transcription of the MHC class II genes, more specifically the domain which is defined by SEQ ID No. 21 of the present invention and which corresponds to the translation of the nucleic acid sequence according to SEQ ID No. 17. Nevertheless, surprisingly and contrary to that which is observed in the case of other factors which are involved in regulating expression of the MHC class II genes (Cogswell et al., 1991, Crit. Rev. Immunol. 11, 87-112), Steimle et al. have demonstrated that expression of the CIITA factor coincides strictly with expression of the MHC class II genes and is required absolutely both for constitutively expressing and for inducing the said MHC genes. Furthermore, Silacci et al. (1994, J. Exp. Med., 180, 1329-1336) have demonstrated that suppression of the MHC class II genes during plasma cell differentiation is associated with suppression of the gene which encodes CIITA factor.
Moreover, Lennon et al. (1997, Immunogenetics, 45, 266-273) have identified the promoter sequence of a CIITA gene, which sequence is responsible for the differential expression of this factor in B cells. However, the existence of this sequence alone does not explain why differential expression of the CIITA factor is observed in different cell types. Furthermore, it does not account for induction by cytokines.
Using samples derived from different tissues of human origin, the applicant has now identified the complex organization of the sequences which ensure regulation of the expression of the CIITA factor, has isolated and characterized other promoter regions and has demonstrated the existence of several forms of CIITA factor, and has also demonstrated the existence of different CIITA genes.
The expression xe2x80x9cCIITA genexe2x80x9d is understood as meaning a nucleic acid sequence which consists of a promoter (P) moiety, an untranslated (UT) moiety and a coding (Prot) moiety, with the coding moiety encoding one of the identified forms of CIITA factor.
More precisely, the inventors have identified a number of nucleic acid sequences which represent CIITA genes and which are therefore capable, in particular, of being involved in controlling and regulating the expression of genes encoding MHC class II molecules. The expression xe2x80x9cnucleic acid sequence which represents CIITA genesxe2x80x9d is understood as meaning that the sequence in question comprises all or part of a nucleic acid sequence corresponding to the mRNAs which derive from the different tissues or cell lines which express CIITA activity either constitutively or following induction. Such sequences can therefore equally well be sequences which are at least partially coding, as for example sequences which are involved in controlling the expression, in particular, of sequences which possess a transcriptional promoter activity.
The expression xe2x80x9cnucleic acid sequencexe2x80x9d is understood as meaning a natural, isolated, or synthetic, double-stranded or single-stranded DNA and/or RNA fragment which designates a precise linked-up series of modified or unmodified nucleotides and which makes it possible to define a fragment or a region of a nucleic acid.
The expression xe2x80x9cpolypeptidexe2x80x9d is understood as meaning a precise, natural, isolated, or synthesized, modified or unmodified linked-up series of amino acids, independently of its size or its function.
The expression xe2x80x9callelic variantxe2x80x9d of a polypeptide is understood as meaning the entirety of the mutated polypeptides and the polymorphisms which can exist in man, and which are obtained, in particular, by truncating, substituting, deleting or adding on amino acid residues, as well as the artificial variants which are employed in vitro.
The expression xe2x80x9cnucleic acid sequence which exhibits a transcriptional promoter activityxe2x80x9d is understood as meaning a nucleic acid sequence which makes it possible to control, that is initiate and/or modulate, the transcription of at least one homologous or heterologous gene which is located downstream of the said sequence. Similarly, reference will be made to the promoter function of the said sequences.
The expression xe2x80x9cnucleic acid sequence which is homologous to a first nucleic acid sequencexe2x80x9d is understood as meaning a nucleic acid sequence which naturally exhibits a functional link with the said first sequence. Thus, according to the invention, a nucleic acid sequence which exhibits a CIITA promoter activity, that is which naturally directs the transcription of a nucleic acid sequence encoding a CIITA factor, is, for example, considered as being homologous to this same nucleic acid sequence which encodes a CIITA gene. In the opposite case, reference will be made to a xe2x80x9cheterologous nucleic acid sequencexe2x80x9d.
The expression xe2x80x9creporter genexe2x80x9d is understood as meaning any nucleic acid sequence which is located downstream of a second nucleic acid sequence and which makes it possible to analyse the transcriptional promoter activity of the said second sequence. Thus, transcription of this reporter gene is manifested by the appearance of a product (RNA or polypeptide) which can readily be detected using well known conventional techniques.
It should be understood that the present invention does not relate to genomic nucleotide sequences in their natural chromosomal environment, that is in the natural state; on the contrary, the sequences are sequences which have been isolated, meaning that they have been directly or indirectly withdrawn, for example by copying (cDNA), and that their environment has been at least partially modified.
The invention thus relates to a nucleic acid sequence which comprises all or part of a nucleic acid sequence of a CIITA gene and which is selected from the sequences SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, and their complementary sequences.
The invention more specifically relates to such a nucleic acid sequence which comprises all or part of a sequence which exhibits a transcriptional promoter activity.
Particularly interesting sequences which may be mentioned are those which comprise all or part of a sequence which is selected from SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6, and their complementary sequences.
The sequences which contain all or part of the sequence identified as SEQ ID No. 4, or its complementary sequence, are particularly advantageous in that they are able to express their transcriptional promoter activity specifically in one cell type, such as dendritic cells.
Certain sequences which have been identified in accordance with the invention are able to express their transcriptional promoter activity following induction by a cytokine such as, for example, interferon xcex3 or interleukin 4. A preferred example of such a sequence is represented by the sequence which comprises all or part of an identified sequence SEQ ID No. 6, or its complementary sequence.
The invention also relates to the nucleic acid sequences which comprise all or part of a sequence selected from:
a) a nucleic acid sequence which encodes a polypeptide which possesses an amino acid sequence such as shown in SEQ ID No. 16, and its complementary sequence,
b) the sequences SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 10 and SEQ ID No. 11 and their complementary sequences,
c) a nucleic. acid sequence which encodes an allelic variant of a polypeptide such as defined in a), or its complementary sequence.
The present invention furthermore relates to a nucleic acid sequence which comprises at least one sequence exhibiting a transcriptional promoter activity, such as, in particular, the sequences which comprise all or part of the sequences SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6, or their complementary sequences, which sequence is located upstream of at least one heterologous or homologous nucleic acid sequence such as, for example, a nucleic acid sequence which comprises all or part of a sequence selected from:
a) a nucleic acid sequence which encodes a polypeptide which consists of the amino acids identified in SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18 or SEQ ID No. 19, or its complementary sequence,
b) the identified nucleic acid sequences is SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14 and SEQ ID No. 15, and their complementary sequences,
c) a nucleic acid sequence which encodes an allelic variant of a polypeptide such as defined in a), or its complementary sequence.
It should be made clear that, in this case, it is possible to have at least two sequences which exhibit a transcriptional promoter activity and/or at least two heterologous or homologous nucleic acid sequences which are situated contiguously or at a distance in relation to each other, and in the same direction or in opposite directions, without the transcriptional promoter function or the transcription of the said sequences thereby being affected.
Similarly, it is possible, in this type of nucleic acid construction, to introduce xe2x80x9cneutralxe2x80x9d nucleic acid sequences or introns which do not affect transcription and which are spliced before the translation step. Sequences of this nature, and their uses, are widely described in the literature.
According to the invention, the nucleic acid sequences, or their fragments, can, in particular, encode all or part of polypeptides which possess the amino acid sequence of a CIITA factor as described in the present invention.
It will then be stated that they encode CIITA polypeptides.
The sequences can also be employed as probes or as primers in processes for detecting or identifying or enzymically amplifying nucleic acid. In this case, the fragments exhibit a minimum size of 10 bases, and preference will be given to fragments of 20 bases, preferably of 30 bases.
The present invention also relates to a nucleic acid sequence which possesses a sequence which is complementary to a target sequence which belongs to a gene or to an RNA whose expression it is desired to block specifically. An antisense oligonucleotide which hybridizes with the sequence to which it is complementary and can thereby block expression of the mRNA having this sequence constitutes such a sequence. In this context, the term xe2x80x9coligonucleotidexe2x80x9d is used in a general manner to designate a polynucleotide of from 2 to 100, more generally of from 5 to 50, ribonucleotides, deoxyribonucloetides or mixed nucleotides in a series. According to the invention, such a sequence is able to hybridize with a nucleic acid sequence which comprises a sequence exhibiting a transcriptional promoter activity or with a nucleic acid sequence comprising a sequence such as previously defined in a), b) or c), and is furthermore able either to block the promoter activity of the said sequence or to inhibit the synthesis of the polypeptide which is encoded by the said sequence.
The hybridization conditions are determined, according to the invention, in order to ensure at least 95% homology. The skilled person is in possession of sufficient knowledge to enable him to define the said conditions.
Even if the described sequences are generally normal sequences, the invention also relates to sequences which are mutated to the extent that they include at least one point mutation and preferably less than 20 mutations in all.
Preferably, the present invention relates to nucleotide sequences in which the point mutations are not silent, that is they lead either to a change in the regulation of the efficacy or of the cellular specificity of the transcription of the gene which is located downstream of the said sequence, or to a change in the coding sequence which affects expression of the CIITA gene, or to a change in the encoded amino acid as compared with the normal sequence, which change affects the function of the corresponding CIITA factor.
The present invention relates, in particular, to a nucleic acid sequence which comprises at least one mutation which affects the transcriptional promoter function of the said sequence. Preferably, these mutations concern the regions which are involved in the transcriptional promoter activity and which make it possible to bind factors which are involved in the transcription initiation, activation or modulation step or in transcription more generally. These regions can, for example, consist of at least one site which is involved in the transcription process and which is selected from the group consisting of the NF-GMb site (Shannon et al., 1988, Proc. Natl. Acad. Sci. USA, 85, 674-678), the NF-IL6 site (Akira and Kishimoto, 1992, Immunil. Rev. 127, 25-50), the PEA3 site (Wasylyk et al., 1989, EMBO J., 8, 3371-3378), the API site (Pollock and Treisman, 1990, Nucleic Acid Res. 18, 6197-6204), the CCAAT box (Dorn et al., 1987, Cell, 50, 863-872), the E2A box (Murre et al., 1989, Cell, 56, 777-783), the IRF1/2 site (Tanaka et al., 1993, Molecular and Cellular Biology, 13, 4531-4538), the MYC site (Agira et al., 1989, EMBO J., 8, 4273-4279), the OCT site (Rosales et al., 1987, EMBO J., 6, 3015-3025), the NF-GMa site (Shannon et al., 1988, Proc. Natl. Acad. Sci. USA, 85, 674-678), the GAS box (Pelligrini and Schindler, 1993, Trends Biochem. Sci., 18, 338-342), the E box (Blackwell et al., 1990, Science, 250, 1149-1151) and the NfxcexaB site (Sen and Baltimore, 1986, Cell, 46, 705-716).
The present invention also relates to a nucleotide sequence which can include unnatural nucleotides, in particular sulphur-containing nucleotides, for example, or nucleotides having an xcex1 or xcex2 structure, or nucleotides which are labelled with a label which is, by way of example, selected from the group consisting of an enzyme, biotin, iminobiotin, a fluorescent compound, a radioactive compound, a chemiluminescent compound, an electrodense compound, a magnetic compound, an antigen, a hapten and an antibody.
The present invention also relates to cloning or expression vectors which include at least one nucleotide sequence such as previously described.
These cloning or expression vectors can additionally include elements which ensure expression of the sequence in the host cell, in particular promoter sequences and/or regulatory sequences which are effective in the said cell, if the sequence is a coding sequence.
If the sequence is a sequence which possesses a transcriptional promoter activity, the vector will additionally include homologous or heterologous nucleic acid sequences which it is desired to express in the said cell.
Preferably, these cloning or expression vectors comprise at least one gene of interest which is placed under the control of at least one nucleic acid sequence which is as previously described and which exhibits a transcriptional promoter activity.
The said gene of interest can, for example, be selected from the group which consists of the genes which encode the CIITA factor and the xcex1 and xcex2 chains of the HLA-DR, HLA-DQ and/or HLA-DP molecules, and reporter genes, such as the gene which encodes rabbit xcex2 globin.
The vector in question can be selected from the autonomously replicating vectors or from the vectors which integrate into the chromosome.
In the case of an autonomously replicating system, use will preferably be made, depending on whether the cell host is prokaryotic or eukaryotic, of systems of the plasmid type or of viral systems, with it being possible for the viral vectors to be, in particular, adenoviruses, poxviruses or herpesviruses. The skilled person is familiar with the techniques which can be used for each of these viruses.
When it is desired to integrate the sequence into the host cell chromosomes, it will be necessary to provide one or more sequences derived from the cell host at each end of the nucleic acid sequence to be integrated in order to ensure recombination. These are also processes which are widely described in the prior literature. Use can, in particular, be made of systems of the plasmid or viral type such as, for example, retroviruses or AAVs (adenoassociated viruses).
The invention also relates to prokaryotic or eukaryotic cells which have been transformed with a vector such as previously described, in particular in order to ensure expression of at least one of the forms of the CIITA factor which have been identified in accordance with the invention.
Cells which can be used for implementing the invention and which may indeed be mentioned are prokaryotic cells, yeast cells and animal cells, in particular cultures of mammalian cells.
Preferably, the host cell is selected from the group consisting of dendritic cells, B lymphocytes, T lymphocytes, macrophages, monocytes, thymus epithelium cells, muscle cells, fibroblasts, endothelial cells and cancer cells, in particular melanoma cells.
The cells which have thus been obtained can be used to prepare natural or mutated CIITA polypeptides and also fragments of these polypeptides.
These cells can also be used as model cells for the purpose of studying the mechanisms of regulating the transcriptional promoter function of the sequences which have been identified in accordance with the invention and of identifying specific inhibitors whose action could possibly be targeted in a given cell type. These cells can additionally be used as model cells for the purpose of studying the interactions between the different CIITA factors which have been isolated, or their variants, and the regions which direct transcription of the genes encoding the MHC class II molecules, and, especially, for the purpose of selecting the variants of the CIITA factors which are able to act as agonists or antagonists on the CIITA receptor. These types of cell model can be constructed using known techniques of genetic manipulation. Furthermore, the use of such cell models with a view to testing pharmaceutical compounds is well known to the skilled person.
The present invention also relates to organisms, such as animals, in particular mice, whose genome has been genetically modified in order to integrate at least one of the nucleic acid sequences according to the invention. In this case, again, these animals can be used as model animals in order to test the efficacy of particular pharmaceutical products.
The present invention also relates to a process for producing a CIITA polypeptide, in particular as defined in SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18 or SEQ ID No. 19, or an allelic variant of one of these polypeptides, which process comprises (i) culturing a host cell, which has been transformed with a vector which includes a nucleic acid sequence encoding a CIITA polypeptide as previously described, under culture conditions which are appropriate for producing the said polypeptide, and (ii) recovering the said polypeptide.
The said polypeptide can be recovered intracellularly or extracellularly in the culture medium when the vector has been designed for assuring secretion of the polypeptide by the expedient, for example, of using a leader sequence, with the polypeptide being in the form of a prepolypeptide. The constructs which enable polypeptides to be secreted are known, both for prokaryotic systems and eukaryotic systems.
The present invention also relates to a CIITA polypeptide which can be obtained by implementing the abovedescribed process.
The present invention additionally relates to CIITA polypeptides which correspond to the previously described nucleic acid sequences and which are in unnatural form, i.e. they are not used in their natural environment but are obtained by purification from natural sources or else obtained by genetic recombination.
More specifically, the invention relates to a polypeptide which is characterized in that it comprises at least one sequence which is selected from:
a) an amino acid sequence which consists of SEQ ID No. 16, or its allelic variants,
b) an amino acid sequence which consists of a part of the sequence SEQ ID No. 16, or its allelic variants.
Preferably, a peptide of this nature is characterized in that at least one part of its sequence is defined in the space between amino acid 1 and amino acid 94 of the sequence SEQ ID No. 16.
The invention also relates to the same polypeptides which are obtained by chemical synthesis and which can include unnatural amino acids. The invention also relates to the said polypeptides which are in a form which is totally or partially retro and/or inverso and which exhibit an activity which is equivalent to that observed in the case of the native CIITA factor, or one of its variants, according to the present invention, or at least an immunological activity which is identical to that of the parent CIITA factor.
Furthermore, the polypeptides, and more specifically their variants, such as previously described, can exhibit the same function of transactivating the expression of the genes encoding MHC class II molecules as a CIITA factor or, at least, the same ability to bind to the specific site for binding a CIITA factor during expression of the said genes.
The present invention additionally relates to an antibody which is directed against any one of the previously described polypeptides or against a polypeptide which contains at least one mutation affecting the function of the CIITA factor, as described below, and, more specifically, to a polyclonal or monoclonal antibody which is obtained by the immunological reaction of a human or animal organism with an immunogenic agent which comprises at least one of the said polypeptides.
The invention also relates to molecules which are able to inhibit either the function of the polypeptides which have been identified according to the invention in activating expression of the genes encoding the MHC class II molecules or the ability of these polypeptides to bind to the CIITA-binding site. These molecules can be polypeptides which contain at least one mutation which affects the function of the CIITA factor. A modified polypeptide of this nature, which consists, for example, of a structural analogue of the said polypeptide, can act as a lure. The molecules can also be antibodies, such as presented above, which are able, for example, to block either all or part of the CIITA factor which is able to react with its specific receptor, or a region of the CIITA factor which is able to interact with at least one other transactivating factor during expression of the genes encoding the MHC class II molecules.
The invention also relates to molecules which are able specifically to inhibit expression of the genes which encode MHC class II molecules in dendritic cells. These molecules consist, in particular, of all or part of a nucleic acid sequence which contains at least one mutation which affects the transcriptional promoter function of the said sequence, with the mutation(s) being located in an identified nucleic acid sequence SEQ ID No. 4, or its complementary sequence.
The invention also relates to molecules which are able specifically to inhibit the induction, by cytokines, of the expression of the genes which encode MHC class II molecules. These molecules consist, in particular, of all or part of a nucleic acid sequence which contains at least one mutation which affects the transcriptional promoter function of the said sequence, with the mutation(s) being located in the identified nucleic acid sequence SEQ ID No. 6, or its complementary sequence.
The present invention also relates to pharmaceutical compositions which comprise, as the active principle, at least one substance such as a nucleic acid sequence or an inhibitory molecule as previously defined. More specifically, the invention relates to a pharmaceutical composition for treating disorders in which it is desired to increase expression of the genes encoding MHC class II molecules, in particular in one cell type, more specifically in a dendritic cell. Furthermore, it is possible to observe this increase in the expression of the genes encoding MHC class II molecules following induction by a cytokine, more specifically by interferon xcex3 or interleukin 4, in particular when the said pharmaceutical composition comprises at least one substance which consists of a nucleic acid sequence which can be activated by the said cytokine, as previously described. The invention additionally relates to a said pharmaceutical composition for treating disorders in which it is desired to reduce expression of the genes encoding MHC class II molecules, more specifically to a pharmaceutical composition which comprises, as the active principle, a) either a nucleic acid sequence according to the invention, the sequence of which is modified such that the promoter activity of the said sequence is affected, or which leads to the production of an inactive CIITA polypeptide, as previously described, or b) an inactive CIITA polypeptide.
The invention furthermore relates to a vaccine which can be used, in particular, for treating cancer or autoimmune diseases, characterized in that it comprises at least one of the pharmaceutical compositions presented above.
Finally, the present invention relates, more specifically, to methods for diagnosing a predisposition to a disorder which is linked to a disturbance in the expression of the genes encoding MHC class II molecules, characterized in that a biological sample is taken from a patient, and the presence of at least one mutation, within either sequences which exhibit a transcriptional promoter activity or sequences which encode one of the identified CIITA factors according to the present invention, is determined by analysing the said nucleic acid sequences and comparing with the wild-type sequences according to the invention, with the presence of at least one such mutation being indicative of a predisposition of the said patient to the said disorder.
A large number of disorders which are directly or indirectly linked to a disturbance in the expression of the genes encoding MHC class II molecules have been described in the literature. We may cite, by way of example, disorders such as insulin-dependent diabetes, multiple sclerosis, rheumatoid arthritis and lupus erythematosus, one of the elements of which disorders could be overexpression of the genes encoding MHC class II molecules; or, conversely, the BLS (bare lymphocytes syndrome) syndrome, which is associated with a severe immunodeficiency.
Sought-after mutations which may be mentioned, more specifically, are mutations which affect the promoter function of nucleic acid sequences, mutations which affect the cellular specificity of the said promoter function, or mutations which affect induction of the said promoter function by a cytokine.
The analysed nucleic acid sequence can equally well be a genomic DNA, a cDNA or an RNA.
The diagnostic tools which are based on the present invention can be used to make a positive and differential diagnosis in a subject taken in isolation or else to make a presymptomatic diagnosis in a subject at risk.
There are, of course, a very large number of methods for demonstrating a mutation in a gene as compared with the natural gene; they can be implemented by studying the genomic DNA, the cDNA, the RNA and/or the polypeptide. They can, essentially, be divided into two broad categories, with the first type of method being that in which the presence of a mutation is detected by comparing the mutated sequence with the corresponding natural, unmutated sequence, and the second type being that in which the presence of the mutation is detected indirectly. Advantageously, the mutation can be detected by demonstrating mismatches, which are due to the presence of the mutation, after analysing by means of hybridization which is carried out using at least one oligonucleotide probe which is specific for the sought-after mutation.
In each of the cases, preference will in general be given to the methods in which all or part of the sequence corresponding to all or part of the identified sequences SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 is amplified prior to demonstrating the mutation. These amplification methods are well known.
Furthermore, the mutated CIITA factors which are found in subjects who are exhibiting disorders in the expression of the genes encoding MHC type II molecules can exhibit an antigenicity which is different from that of the identified natural CIITA factors SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18. It is therefore possible to diagnose or prognosticate susceptibility to disorders which are linked to deregulation of the expression of the genes encoding MHC type II molecules by demonstrating the presence of the product of the mutated CIITA gene, for example using antibodies, in particular monoclonal antibodies, as previously described.
Other characteristics and advantages of the present invention will be apparent from reading the following examples, which are illustrated by FIGS. 1 to 9. Nevertheless, the invention is not limited to the contents of the said examples.